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Doctoral Thesis
DOI
10.11606/T.46.2018.tde-18092018-154812
Document
Author
Full name
Celio Xavier da Costa dos Santos
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2002
Supervisor
Committee
Augusto, Ohara (President)
Campa, Ana
Di Mascio, Paolo
Kowaltowski, Alícia Juliana
Laurindo, Francisco Rafael Martins
Title in Portuguese
Oxidação de urato e tirosina por peroxinitrito. implicações para o desenvolvimento de sequestradores e biomarcadores de peroxinitrito
Keywords in Portuguese
Ácidos nucleicos (Metabolismo)
Aminoácidos (Metabolismo)
Biomarcadores (Desenvolvimento)
Bioquímica orgânica
Abstract in Portuguese
Peroxinitrito (ONOO- + ONOOH), o produto da rápida reação do óxido nítrico com o ânion radical superóxido, tem recebido muita atenção como possível mediador dos efeitos deletérios associados a uma superprodução de NO. O peroxinitrito é um potente oxidante que é capaz de oxidar e nitrar várias biomoléculas por mecanismos que contribuímos para esclarecer no decorrer desta tese. Especificamente, estudamos a oxidação de urato e tirosina por peroxinitrito. Demonstramos que o urato é oxidado por peroxinitrito a alantoina, aloxana e ao radical aminocarbonila. Como a reação direta entre urato e peroxinitrito tem uma constante de velocidade relativamente baixa (k= 4,8 x 102 M -1.s-1) em comparação com àquelas de outras biomoléculas, sugerimos que o urato é um potente sequestrador dos radicais derivados do peroxinitrito (NO2 e CO3•-, na maioria dos ambientes biológicos; a pH ácido, o radical OH também pode se tornar relevante). No caso da tirosina, confirmamos que ela não reage diretamente com o peroxinitrito mas com os radicais dele derivados. Como antecipado, o rendimento relativo dos produtos (3-nitrotirosina, 3,3-bitirosina e 3-hidroxitirosina (DOPA)) variou com o pH e a presença de CO2. Esses estudos nos levaram a propor a co-localização de proteínas nitradas e hidroxiladas como um possível biomarcador de peroxinitrito. Para testar essa hipótese, um anticorpo monoclonal anti-DOPA foi desenvolvido e utilizado em modelos de infecção por Leishmania amazonenses (macrófagos (J774), e camundongos resistentes (C56Bl/6) e suscetíveis (BALB/c). A co-localização de proteínas hidroxiladas e nitradas ficou evidênciada em todos os modelos testados e ocorreu concomitantemente a máxima produção de NO. Infelizmente, o anticorpo obtido perdeu a atividade e ainda não pudemos confirmar esses dados.
Title in English
Oxidation of urate and tyrosine by peroxynitrite implications for the development of sequesters and peroxynitrite biomarkers
Keywords in English
Amino acids (Metabolism)
Biomarkers (Development)
Nucleic acids (Metabolism)
Organic biochemistry
Abstract in English
Peroxynitrite (ONOO- + ONOOH), which is formed by the fast reaction between nitric oxide and superoxide anion, has been receiving increasing attention as a mediator of the deleterious effects associated with an overproduction of NO. The compound is a strong oxidant that is able to oxidize and nitrate a variety of biotargets by mechanisms that this work has contributed to establish. Specifically, we studied the oxidation of urate and tyrosine by peroxynitrite. Urate oxidation produced allantoin, alloxan and the amiocarbonyl radical. Since the rate constant of the direct reaction between urate and peroxynitrite (k= 4,8 x 102 M-1.s-1) is low in comparison with those of other biotargets, we proposed that urate is an efficient scavenger of peroxynitrite-derived radicals (NO2 and CO3•- in most biological environments; at acid pH, the OH radical may also become relevant). ln the case of tyrosine, we confirmed that it does not react directly with peroxynitrite but, instead, with the radicals derived form it. As anticipated, the relative yield of the products (3-nitrotyrosine, 3,3-bityrosine and 3-hydroxytyrosine (DOPA)) varied with the pH and CO2 presence. These results led us to propose that co-localization of nitrated and hydroxylated proteins could be a peroxynitrite biomarker. To test this hypothesis, a monoclonal anti-DOPA antibody was developed and tested in Leishamnia amazonensis infection models (macrophages (J774), and resistant (C56Bl/6) and susceptible mice (BALB/c). It was possible to evidence co-localization of hydroxylated and nitrated proteins in all tested models in a time when NO synthesis was maximum. Unfortunetly, we were unable to confirm these results due to antibody inactvation; new antibody baches are being obtained.
 
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Publishing Date
2018-09-18
 
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